T-bet polymorphisms are associated with asthma and airway hyperresponsiveness

Transcription

1 T-bet polymorphisms are associated with asthma and airway hyperresponsiveness Benjamin A. Raby, Eun-Sook Hwang, Kristel Van Steen, Kelan Tantisira, Stanford Peng, Augusto Litonjua, Ross Lazarus, Cosmas Giallourakis, John D. Rioux, David Sparrow, Edwin K. Silverman, Laurie H. Glimcher, Scott T. Weiss Online Data Supplement

2 Supplemental Methods: Populations: Childhood Asthma Management Program The Childhood Asthma Management Program (CAMP) is a multicentered North American clinical trial designed to investigate the long-term effects of inhaled anti-inflammatory medications in children with mild to moderate asthma (1, 2). Of the 1,041 children enrolled in the original clinical trial, 968 children and 1,518 of their parents contributed DNA samples. 580 complete nuclear families (including 55 families with additional affected offspring) are included in the analysis presented here: 471 of non-hispanic Caucasian descent, 64 of African- American descent, and 47 of Hispanic descent. For inclusion, children required a diagnosis of asthma, which was based on methacholine hyperreactivity (PC 20 no greater than 12.5 mg/ml) and one or more of the following criteria for at least six months in the year prior to recruitment: (i) asthma symptoms at least two times per week; (ii) at least two usages per week of an inhaled bronchodilator; and (iii) daily asthma medication. Children with evidence of severe asthma were excluded. Phenotypic data were collected at baseline and during the course of the clinical trial as previously described (1, 2). Pre- and post-bronchodilator spirometry was performed according to American Thoracic Society recommendations with a volumedisplacement spirometer, and airway responsiveness was assessed by methacholine challenge with the Wright nebulizer tidal breathing technique (1). Total serum IgE was measured by radioimmunosorbent assays from blood samples collected during the screening sessions of the CAMP study and at year four follow up. Of the 592 families of Caucasian, African American

3 or Hispanic descent available for analysis, 12 were removed from family-based analyses due to unreliable parental genotypes at numerous genomic markers, and are not included here. Normative Aging Study The Normative Aging Study (NAS) is a longitudinal study of aging established by the Veterans Administration in 1961 (3). The initial cohort consisted of 2,280 men from the Greater Boston area ages years of age at the time of entry into the study between 1961 and Since entry, volunteers have reported for periodic examinations that include spirometric tests. Nonspecific airway responsiveness has been assessed by a methacholine challenge test since April We use a slightly modified version of the Chatham protocol (4), which has been shown to effectively differentiate asthmatics from normal subjects and to correlate well with the standard method of Chai and colleagues (5) The Chatham protocol consists of one and four breaths of a 5-mg/ml methacholine solution followed by one and four breaths of a 25-mg/ml methacholine solution from a DeVilbiss 646 nebulizer. Our modification of this protocol uses, in addition, an initial five breaths of saline solution and one breath of a 1-mg/ml methacholine solution. After each inhalation level, spirometry is performed at 30, 90, and 180 seconds. All inhalations are 6-second vital capacity inhalations followed by 2 seconds of breath holding. The test is terminated before completion of the dose schedule if FEV 1 declines by 20% or more from the value after saline inhalation.methacholine responders were identified by a fall in FEV 1 20% from baseline during the challenge test. Nonresponders demonstrated < 20% fall in FEV 1. DNA for 200 responders and 436 nonresponders was available for genotyping. At the periodic exams, phlebotomy was performed. Absolute eosinophil counts were performed by a trained technician using a

4 hemcytometer after staining with an aliquot of blood with the Unopette reagent system (Becton Dickinson, Rutherford, NJ). Total serum IgE concentration (IU/ml) was determined by paper radioimmunosorbent test (Pharmacia Diagnostics, Piscataway, NJ), and the mean of two determinations was used for analysis. Allergy skin test reactivity was also assessed as previously described by the prick method of Pepys (6). The following skin tests were performed (with allergens obtained from Hollister-Stier Laboratories, Spokane, WA): ragweed (1:20 weight/volume) mixed trees (1:20), mixed grasses (1:20), and house dust (1:10); histamine phosphate (2.75 mg/ml) as the positive control, and a glycerin control. Human Subjects Informed assent and consent were obtained from the study participants and their parents to collect DNA for genetic studies in CAMP. The Institutional Review Board of the Brigham and Women s Hospital (BWH IRB), as well as those of the other CAMP study centers, approved the studies. BWH IRB approval was obtained for DNA collection in the NHS as well. The genetic studies for NAS were approved by the Partners Healthcare Human Research Committee. Informed consent was obtained from the NAS participants for their regularly scheduled visits. Genetic studies in the Normative Aging Study (NAS) were approved by the Partners Healthcare Human Research Committee and the IRB of the Veterans Administration Hospitals using anonymized data sets. Polymorphism discovery The T-bet locus was resequenced in 30 Caucasian subjects (22 with asthma, 8 without) with dye-terminator dideoxy sequencing chemistry (PE Biosystems, Foster City, CA). This sample size was sufficiently large to detect 89% of variants with at least 5% minor allele frequency in

5 the non-asthmatic samples. Primers and reaction protocols are available on our website ( Sequencing products were analyzed with the ABI 3100 Sequence Detector (Applied Biosystems, Foster City, CA). Chromatograms were analyzed with Phred/Phrap/Consed (7, 8). Variants were identified with Polyphred and by manual review (9). The 8 Caucasian subjects without a diagnosis of asthma were resequenced by similar methods at the Whitehead Institute (JR, CG, SP). Polymorphism genotyping SNPs were genotyped using unlabeled minisequencing reactions and mass spectrometry analysis as implemented in the SEQUENOM platform (Sequenom, San Diego, CA), and by the 5 3 exonuclease assay as implemented in the TaqMAN assay (PE Biosystems, Foster City, CA) (10). We were able to develop reliable genotype assays for 12 of the 24 identified T-bet variants and for four additional variants identified from the public database dbsnp and from previous reports: g C>A (rs ), His33Gln (rs ), Pro485Pro, and g A>G (rs ). Protocol details and primer data are available at Quality control was assessed by several methods. Duplicate genotyping was performed on approximately 10% of the sample to assess genotype reproducibility, with discordancy of less than 1% between runs. Genotype completion rates were greater than 90% for all SNPs, with an average rate of 95.8% completion. Low rates of non-mendelian inheritance were observed for all markers (<<1% for each marker) in the family-based data.

6 Statistical Analysis Parental (in CAMP) and control (in NAS) genotype data were assessed for Hardy-Weinberg equilibrium (HWE) and pairwise linkage disequilibrium (LD). HWE was tested at each SNP locus by an exact method (11). LD was evaluated by a maximum likelihood method (12) to infer phase for dual heterozygotes and was expressed as r 2 (13). Haplotype block structure analysis was performed with Haploview (14). Blocks were defined by the Gabriel definition (D > 0.9, minimum allele frequency 5%) (15). FBAT v1.5.3 (16, 17) was used for SNP association analysis with asthma. Quantitative trait analysis was performed with principal components analysis as implemented in PBAT (FBAT- PC) (18, 19) for the following intermediate phenotypes, using all available measurements: post-bronchodilator FEV 1, airway hyperresponsiveness to methacholine (log-transformed PC 20 ), log-transformed total serum IgE levels, and log-transformed blood eosinophil levels. FBAT-PC models the contribution of genetic loci to the total heritability of the trait over repeated measures, thereby maximizing power to detect evidence of association. For each trait tested, associations were rank-ordered based on power to detect association as determined by the PBAT screening method, and a rank-ordered Bonferroni correction was applied to test for multiple comparisons. Evidence for haplotype-block association was assessed with the likelihood-ratio score test implemented in TRANSMIT (20) for asthma and with FBAT (21) for the quantitative traits. To minimize the number of tests performed, we first screened for evidence of global haplotype association using omnibus test statistics (the global chi-square test in TRANSMIT for asthma association, and the multi-allelic haplotype test in FBAT).

7 Only those blocks with significant global tests were then tested for individual haplotype association. The NAS data was analyzed with SAS (SAS Institute, Carey NC) using the PROC CASECONTROL procedure for comparing responders and nonresponders and using generalized linear models for the quantitative analysis of methacholine responsiveness (logtransformed dose-response slope). Genotypes were coded 0 for common allele homozygotes, 1 for heterozygotes, and 2 for rare allele homozygotes. Models were adjusted for age, pack-years of cigarette smoking, and current smoking status. Additional adjustment for baseline FEV 1 was also assessed.

8 Legend for figures: Figure E1: Single nucleotide polymorphisms in T-bet Physical position of genomic region on chromosome 17q21 (in bases from pter) according to July 2003 Human Genome Browser ( Exon-intron structure of T-bet provided, with grey exonic regions denoting the highly conserved T-BOX DNA-binding domain. Plotted below the gene are the cross-species phylogenetic conservation measures based on a phylogenetic hidden Markov model (phylo-hmm) of alignments of the human Jul (hg16), chimpanzee Nov (pantro1), mouse Feb (mm3), rat Jun (rn3), and chicken Feb (galgal2) assemblies. SNPs underlined were genotyped in CAMP. *denotes SNPs not observed in sequencing panel but reported in public databases and the published literature. Figure E2: Pairwise LD plots (r 2 ) for 16 variants genotyped in CAMP African American (a) and Hispanic (b) parents. Arrow denotes relative position of T-bet gene (not to scale).

9 Figure E1: Single nucleotide polymorphisms in T-bet

10 Figure E2 a: African American b: Hispanic

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